Generation of ordered protein assemblies using rigid three-body fusion

Abstract

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Designed proteins assemble antibodies into modular nanocages

“…We used a computational protocol that rapidly samples all possible fusions from our building block library to identify those with the net rigid body transforms required to generate dihedral, tetrahedral, octahedral, and icosahedral AbCs (14,15). To describe the final nanocage architectures, we follow a naming convention which summarizes the point group symmetry and the cyclic symmetries of the building blocks (16).…”

“…We set out to design proteins that drive the assembly of arbitrary antibodies into symmetric assemblies with well-defined structures. Previous design efforts have successfully built nanocages by computationally fusing (14,15) or docking together (16,17) protein building blocks with cyclic symmetry so that the symmetry axes of the building blocks align with a larger target architecture. For example, an I52 icosahedral assembly is built by bringing together a pentamer and a dimer that align to the icosahedral five-and two-fold symmetry axes, respectively.…”

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

“…Our general rigid helix-fusion based pipeline fulfills the promise of early proposals 16,35 in providing a robust and accurate procedure for generating large protein assemblies by fusing symmetric building blocks and avoiding interface design, and should streamline assembly design for applications in vaccine development, drug delivery and biomaterials more generally. The set of structures generated here goes considerably beyond our previous work with rigid helical fusions 18 , and the "WORMS" software introduced here is quite general and readily configurable to different nanomaterial design challenges. WORMS can be easily extended to other symmetric assemblies including 2D arrays and 3D crystals, and should be broadly useful for generating a wide range of protein assemblies.…”

“…All genes were cloned into E. coli cells (BL21 Lemo21 (DE3)) for expression, using auto-induction 38 at 18° or 37°C for [16][17][18][19][20][21][22][23][24] hours in 500mL scale. Post-induction, cultures were centrifuged at 8,000xG for 15 minutes.…”

“…An alternative approach which avoids the need for designing new interfaces is to fuse oligomeric protein building blocks with helical linkers; while this has led to a number of new materials 15 , lack of rigidity has made the structures of these assemblies difficult to precisely specify. More rigid junctions created by overlapping ideal helices and designing around the junction region has resulted in more predictable structures 16,17 , including closed ring dihedral structures which require even more precise structure predictions 18 . This rigid fusion method, however, has its own set of challenges in comparison to designing a new non-covalent protein-protein interface: first, for any pair of protein building blocks, there are far fewer positions for rigid fusion than are for unconstrained protein-protein docking limiting the space of possible solutions, and second, while in the non-covalent protein interface case the space searched can be limited by restricting building blocks to the symmetry axes of the desired nanomaterial, this is not possible in the case of rigid fusions, making the search more difficult as the number of building blocks increases.…”